Tubulin-binding Domain Research Articles

HURP binding to the vinca domain of β-tubulin accounts for cancer drug resistance

Vinca alkaloids, a class of tubulin-binding agent, are widely used in treating cancer, yet the emerging resistance compromises their efficacy. Hepatoma up-regulated protein (HURP), a microtubule-associated protein displaying heightened expression across various cancer types, reduces cancer cells’ sensitivity to vinca-alkaloid drugs upon overexpression. However, the molecular basis behind this drug resistance remains unknown. Here we discover a tubulin-binding domain within HURP, and establish its role in regulating microtubule growth. Cryo-EM analysis reveals interactions between HURP’s tubulin-binding domain and the vinca domain on β-tubulin -- the site targeted by vinca alkaloid drugs. Importantly, HURP competes directly with vinorelbine, a vinca alkaloid-based chemotherapeutic agent, countering microtubule growth defects caused by vinorelbine both in vitro and in vivo. Our findings elucidate a mechanism driving drug resistance in HURP-overexpressing cancer cells and emphasize HURP tubulin-binding domain’s role in mitotic spindle assembly. This underscores its potential as a therapeutic target to improve cancer treatment.

FGF13 enhances the function of TRPV1 by stabilizing microtubules and regulates acute and chronic itch.

Itching is an aversive somatosensation that triggers the desire to scratch. Transient receptor potential (TRP) channel proteins are key players in acute and chronic itch. However, whether the modulatory effect of fibroblast growth factor 13 (FGF13) on acute and chronic itch is associated with TRP channel proteins is unclear. Here, we demonstrated that conditional knockout of Fgf13 in dorsal root ganglion neurons induced significant impairment in scratching behaviors in response to acute histamine-dependent and chronic dry skin itch models. Furthermore, FGF13 selectively regulated the function of the TRPV1, but not the TRPA1 channel on Ca2+ imaging and electrophysiological recordings, as demonstrated by a significant reduction in neuronal excitability and current density induced by TRPV1 channel activation, whereas TRPA1 channel activation had no effect. Changes in channel currents were also verified in HEK cell lines. Subsequently, we observed that selective modulation of TRPV1 by FGF13 required its microtubule-stabilizing effect. Furthermore, in FGF13 knockout mice, only the overexpression of FGF13 with a tubulin-binding domain could rescue TRP channel function and the impaired itch behavior. Our findings reveal a novel mechanism by which FGF13 is involved in TRPV1-dependent itch transduction and provide valuable clues for alleviating pathological itch syndrome.

Abstract 459: Targeting the non-junctional tumorigenic function of connexin43 in cancer cell plasticity

Abstract Tumors are highly heterogeneous and dynamic, with both cancer cells and cancer stem cells (CSCs) displaying a high degree of plasticity contributing to metastasis and tumor recurrence following treatment. Epithelial-mesenchymal transition (EMT) enables cancer cells to acquire migratory and invasive characteristics, access the circulatory system, and metastasize. Moreover, EMT is associated with therapeutic resistance and CSC generation. Our previous research demonstrated a redistribution of the gap junction protein connexin43 (Cx43) from the plasma membrane to the cytosol during EMT. Using super-resolution STochastic Optical Reconstruction Microscopy (STORM), we have recently identified increased interaction of such intracellular Cx43 with microtubules during EMT and in primary glioblastoma (GBM) CSCs (GSCs). We further report a tumorigenic function for cytosolic Cx43/microtubule complexing in cancer cell and CSC proliferation, migration, and survival. Cx43 localization and activity is regulated by multiple sites for protein-protein interaction within the Cx43 carboxy-terminus (CT), including a tubulin binding domain. We employed a Cx43 mimetic peptide named JM2 (juxtamembrane 2) encompassing the microtubule binding sequence of the Cx43 CT. Following administration, JM2 efficiently and specifically disrupts the interaction of Cx43 with microtubules, inhibits GSC-derived neurosphere formation in vitro, and patient GBM-derived organoid growth ex vivo. Using an orthotopic GBM patient derived xenograft mouse model, we demonstrate in vivo that JM2 significantly decreases the number of GSCs within brain tumors, inhibits the formation of highly invasive GBM tumors, and increases mouse survival in combination with the chemotherapy alkylating agent temozolomide. Further examining the role of Cx43/microtubule complexing in the context of metastatic disease, we observed JM2 also decreases mesenchymal marker expression and inhibits cell migration and invasion in EMT models. Using an in vivo orthotopic breast cancer mouse model we find that intratumoral administration of JM2 prevents metastasis, significantly decreasing cancer cell lung colonization. Our findings validate targeting Cx43 complexing with microtubules, with JM2, as a novel therapeutic approach for cancer treatment through inhibition of EMT-associated increases in invasion and metastasis, and elimination of CSCs. Modulation of non-junctional Cx43 functions therefore represents a viable therapeutic approach to target cellular plasticity with broad therapeutic implications for many cancers. Citation Format: Samy Lamouille, Christina E. Wheeler, Stacie E. Deaver, Michael J. Zeitz, Zhi Sheng, Robert G. Gourdie, James W. Smyth. Targeting the non-junctional tumorigenic function of connexin43 in cancer cell plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 459.

Abstract 959: Non-junctional connexin43 complexing with microtubules promotes epithelial-mesenchymal transition

Abstract Epithelial-mesenchymal transition (EMT) is a normal process of embryonic development, stem cell differentiation, and wound healing. During EMT, epithelial cells lose apical-basal polarity, remodel intercellular junctions, and acquire migratory and invasive abilities as they become mesenchymal. Pathologically, EMT has been demonstrated to contribute to intravasation of metastatic tumor cells into the vasculature. Among the activators of EMT, TGF-β has been shown to be most prevalent and is sufficient for experimental induction of EMT. Gap junctions, comprising connexin proteins, allow direct mechanical and metabolic coupling between adjacent cells, and represent a major intercellular structure altered during EMT. Connexin43 (Cx43) is a gap junction protein that has been implicated as both a tumor suppressor and enhancer depending on cancer stage. Our previous work revealed that despite losses in Cx43 gap junctions during EMT, mRNA and cytosolic levels of Cx43 protein increased. Such a shift of Cx43 to cytoplasmic pools has previously been observed in cancer progression and research suggests that non-junctional Cx43 can regulate the cytoskeleton, including microtubule stability. The C-terminus of Cx43 encompasses a tubulin binding domain which, while implicated in Cx43 trafficking, remains functionally poorly understood. We have identified a tumorigenic role for non-junctional Cx43 through its interaction with microtubules in glioma cancer stem cells. This led us to hypothesize that increased cytosolic Cx43 during EMT promotes Cx43-microtubule interaction to facilitate mobility and invasion of metastatic cancer cells. Using several independent TGF-β-induced EMT models, we observe increased complexing of Cx43 and microtubules during EMT by super-resolution microscopy. To study the role of Cx43 interaction with microtubules during EMT, we generated Cx43 harboring mutations in the tubulin binding domain which were ectopically expressed by adenoviral transduction in Cx43 knockout CRISPR/Cas9 epithelial cells. We find these Cx43 mutants fail to interact with microtubules compared to wild-type Cx43. Turning to TGF-β-induced EMT, ablation of the Cx43/microtubules interaction with these mutants is sufficient to decrease expression of mesenchymal markers and inhibit cell migration. We are currently investigating the effects of disrupting Cx43/microtubule complexing in vivo with a breast cancer mouse model to further elucidate effects on tumor progression and metastasis. Given the complex relationship between connexins and cancer progression, our findings support targeting this pathological ‘non-junctional’ Cx43 population specifically to limit metastatic disease, as Cx43 gap junction function is critical to normal tissue function and contributes to tumor suppression. Citation Format: Aryo Sorayya, Christina Wheeler, Stacie Deaver, Kenneth Young, Michael Zeitz, James Smyth, Samy Lamouille. Non-junctional connexin43 complexing with microtubules promotes epithelial-mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 959.

The evolution of microtubule associated proteins \u2013 a reference proteomic perspective

Microtubule associated proteins (MAPs), defined as proteins that bind microtubules but are not molecular motors or severing enzymes, play a key role in regulating microtubule stability in neurons. Existing studies of the evolutionary relationships between these proteins are limited to genomic data from a small number of species. We therefore used a large collection of publicly available reference-quality eukaryotic proteomes to carry out a phylogenetic analysis of microtubule associated proteins in both vertebrates and invertebrates. Complete or near-complete reference quality proteomes were obtained from Uniprot. Microtubule associated proteins were identified using InterProtScan, aligned using MUSCLE and then phylogenetic trees constructed using the WAG algorithm. We identified 889 proteins with tubulin binding domains, of which 663 were in eukaryotes, including 168 vertebrates and 64 invertebrates. The vertebrate proteins separated into three families, resembling human MAP 2, MAP4 and MAPT, respectively, while invertebrate MAPs clustered separately. We found significant variation in number of microtubule associated proteins and number of microtubule binding domains between taxa, with fish and mollusks having an unexpectedly high number of MAPs and binding domains, respectively. Our findings represent a novel analysis of the evolution of microtubule associated proteins based on publicly available proteomics data sets. We were able to confirm the phylogeny of MAPs identified based on more limited genomic analyses, and in addition, derived several novel insights on the structure and function of MAPs.

FGF13 enhances resistance to platinum drugs by regulating hCTR1 and ATP7A via a microtubule-stabilizing effect.

Platinum‐based regimens are the most widely used chemotherapy regimens, but cancer cells often develop resistance, which impedes therapy outcome for patients. Previous studies have shown that fibroblast growth factor 13 (FGF13) is associated with resistance to platinum drugs in HeLa cells. However, the mechanism and universality of this effect have not been clarified. Here, we found that FGF13 was associated with poor platinum‐based chemotherapy outcomes in a variety of cancers, such as lung, endometrial, and cervical cancers, through bioinformatics analysis. We then found that FGF13 simultaneously regulates the expression and distribution of hCTR1 and ATP7A in cancer cells, causes reduced platinum influx, and promotes platinum sequestration and efflux upon cisplatin exposure. We subsequently observed that FGF13‐mediated platinum resistance requires the microtubule‐stabilizing effect of FGF13. Only overexpression of FGF13 with the ‐SMIYRQQQ‐ tubulin‐binding domain could induce the platinum resistance effect. This phenomenon was also observed in SK‐MES‐1 cells, KLE cells, and 5637 cells. Our research reveals the mechanism of FGF13‐induced platinum drug resistance and suggests that FGF13 can be a sensibilization target and prognostic biomarker for chemotherapy.

A cryptic tubulin-binding domain links MEKK1 to curved tubulin protomers

The MEKK1 protein is a pivotal kinase activator of responses to cellular stress. Activation of MEKK1 can trigger various responses, including mitogen-activated protein (MAP) kinases, NF-κB signaling, or cell migration. Notably, MEKK1 activity is triggered by microtubule-targeting chemotherapies, among other stressors. Here we show that MEKK1 contains a previously unidentified tumor overexpressed gene (TOG) domain. The MEKK1 TOG domain binds to tubulin heterodimers-a canonical function of TOG domains-but is unusual in that it appears alone rather than as part of a multi-TOG array, and has structural features distinct from previously characterized TOG domains. MEKK1 TOG demonstrates a clear preference for binding curved tubulin heterodimers, which exist in soluble tubulin and at sites of microtubule polymerization and depolymerization. Mutations disrupting tubulin binding decrease microtubule density at the leading edge of polarized cells, suggesting that tubulin binding may play a role in MEKK1 activity at the cellular periphery. We also show that MEKK1 mutations at the tubulin-binding interface of the TOG domain recur in patient-derived tumor sequences, suggesting selective enrichment of tumor cells with disrupted MEKK1-microtubule association. Together, these findings provide a direct link between the MEKK1 protein and tubulin, which is likely to be relevant to cancer cell migration and response to microtubule-modulating therapies.

A doublecortin-domain protein of Toxoplasma and its orthologues bind to and modify the structure and organization of tubulin polymers

BackgroundTgDCX is a doublecortin-domain protein associated with the conoid fibers, a set of strongly curved non-tubular tubulin-polymers in Toxoplasma. TgDCX deletion impairs conoid structure and parasite invasion. TgDCX contains two tubulin-binding domains: a partial P25α and the DCX/doublecortin domain. Orthologues are found in apicomplexans and their free-living relatives Chromera and Vitrella.ResultsWe report that isolated TgDCX-containing conoid fibers retain their pronounced curvature, but loss of TgDCX destabilizes the fibers. We crystallized and determined the 3D-structure of the DCX-domain, which is similar to those of human doublecortin and well-conserved among TgDCX orthologues. However, the orthologues vary widely in targeting to the conoid in Toxoplasma and in modulating microtubule organization in Xenopus cells. Several orthologues bind to microtubules in Xenopus cells, but only TgDCX generates short, strongly curved microtubule arcs. EM analysis shows microtubules decorated with TgDCX bundled into rafts, often bordered on one edge by a “C”-shaped incomplete tube. A Chromera orthologue closely mimics TgDCX targeting in Toxoplasma and binds to microtubules in Xenopus cells, but does not generate arcs or “C”-shaped tubes, and fails to rescue the defects of the TgDCX-knockout parasite.ConclusionsThese observations suggest that species-specific features of TgDCX enable it to generate strongly curved tubulin-polymers to support efficient host-cell invasion.

Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer

Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer

Independent tubulin binding and polymerization by the proline-rich region of Tau is regulated by Tau's N-terminal domain

Tau is an intrinsically disordered, microtubule-associated protein that has a role in regulating microtubule dynamics. Despite intensive research, the molecular mechanisms of Tau-mediated microtubule polymerization are poorly understood. Here we used single-molecule fluorescence to investigate the role of Tau's N-terminal domain (NTD) and proline-rich region (PRR) in regulating interactions of Tau with soluble tubulin. We assayed both full-length Tau isoforms and truncated variants for their ability to bind soluble tubulin and stimulate microtubule polymerization. We found that Tau's PRR is an independent tubulin-binding domain that has tubulin polymerization capacity. In contrast to the relatively weak interactions with tubulin mediated by sites distributed throughout Tau's microtubule-binding region (MTBR), resulting in heterogeneous Tau: tubulin complexes, the PRR bound tubulin tightly and stoichiometrically. Moreover, we demonstrate that interactions between the PRR and MTBR are reduced by the NTD through a conserved conformational ensemble. On the basis of these results, we propose that Tau's PRR can serve as a core tubulin-binding domain, whereas the MTBR enhances polymerization capacity by increasing the local tubulin concentration. Moreover, the NTD appears to negatively regulate tubulin-binding interactions of both of these domains. The findings of our study draw attention to a central role of the PRR in Tau function and provide mechanistic insight into Tau-mediated polymerization of tubulin.

GCN5L1 interacts with αTAT1 and RanBP2 to regulate hepatic α-tubulin acetylation and lysosome trafficking.

Although GCN5L1 (also known as BLOC1S1) facilitates mitochondrial protein acetylation and controls endosomal-lysosomal trafficking, the mechanisms underpinning these disparate effects are unclear. As microtubule acetylation modulates endosome-lysosome trafficking, we reasoned that exploring the role of GCN5L1 in this biology may enhance our understanding of GCN5L1-mediated protein acetylation. We show that α-tubulin acetylation is reduced in GCN5L1-knockout hepatocytes and restored by GCN5L1 reconstitution. Furthermore, GCN5L1 binds to the α-tubulin acetyltransferase αTAT1, and GCN5L1-mediated α-tubulin acetylation is dependent on αTAT1. Given that cytosolic GCN5L1 has been identified as a component of numerous multiprotein complexes, we explored whether novel interacting partners contribute to this regulation. We identify RanBP2 as a novel interacting partner of GCN5L1 and αTAT1. Genetic silencing of RanBP2 phenocopies GCN5L1 depletion by reducing α-tubulin acetylation, and we find that RanBP2 possesses a tubulin-binding domain, which recruits GCN5L1 to α-tubulin. Finally, we find that genetic depletion of GCN5L1 promotes perinuclear lysosome accumulation and histone deacetylase inhibition partially restores lysosomal positioning. We conclude that the interactions of GCN5L1, RanBP2 and αTAT1 function in concert to control α-tubulin acetylation and may contribute towards the regulation of cellular lysosome positioning. This article has an associated First Person interview with the first author of the paper.

Abstract 4765: Targeting glioblastoma cancer stem cells with a novel Connexin43 mimetic peptide

Abstract Glioblastoma (GBM) is a highly malignant and lethal cancer of the central nervous system. Despite intensive research efforts, there has been limited progress in improving patient outcome. The current therapy for GBM patients includes surgical resection, radiotherapy and cytotoxic chemotherapy with temozolomide (TMZ), conferring a median survival time of only 14.6 months. Failure to generate more effective treatment strategies is due in part to the cellular heterogeneity within GBM tumors, which comprise a sub-population of GBM cancer stem cells (GSCs) characterized by self-renewal characteristics and resistance to TMZ. Recent work, including our own, has demonstrated that levels of the gap junction protein Connexin43 (Cx43) correlate with GBM TMZ resistance. Increased Cx43 levels occur in GSCs compared to parental GBM cells, and patients with high levels of Cx43 mRNA and low levels of MGMT, an enzyme that repairs TMZ-induced DNA lesions, have a significantly shorter life span. In contrast, Cx43 has also been associated with anti-proliferative effects in glioma and reduced levels of Cx43 protein are reported in high-grade gliomas. In addition to forming gap junctions, Cx43 can regulate cell proliferation, migration, and apoptosis, through distinct channel-independent mechanisms. Therefore, altering the localization and/or activity of Cx43 rather than Cx43 expression alone may represent a more targeted strategy in GBM treatment. Using super-resolution microscopy, we find intracellular Cx43 decorating microtubules in GSCs demonstrating for the first time such clustering in situ. Regulation of Cx43 function is primarily associated with multiple sites for post-translational modification and protein-protein interaction within the Cx43 carboxy-terminus (CT) which includes a tubulin binding domain. We have developed a peptide named JM2 (juxtamembrane 2) composed of the Cx43 CT amino acids encompassing the microtubule-binding sequence fused to an antennapedia cell penetration domain for cellular uptake. Super-resolution microscopy and biochemical analysis confirm JM2 specific interaction with microtubules concomitantly with a loss of Cx43 interaction with microtubules in GSCs derived from patient tumors. In addition, we observe that JM2 decreases Cx43 gap junction plaque formation and cell-cell communication in GSCs and limits microtubule dynamics. Importantly, JM2 decreases cell survival in TMZ-resistant GSCs and GSC neurosphere formation in vitro, and GSC-derived tumor growth in vivo. Our current research includes the development of JM2-loaded biodegradable nanoparticles for sustained JM2 delivery in preparation for future clinical trials. In conclusion, we have developed a Cx43 mimetic peptide that significantly decreases the tumorigenic potential of GSCs and represents a novel and potent therapeutic opportunity to alter Cx43 activity in targeting chemoresistant GSCs for GBM treatment. Citation Format: Samy Lamouille, James W. Smyth, Laurie O'Rourke, Pratik Kanabur, Sujuan Guo, Jane Jourdan, Zhi Sheng, Robert G. Gourdie. Targeting glioblastoma cancer stem cells with a novel Connexin43 mimetic peptide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4765. doi:10.1158/1538-7445.AM2017-4765

Cadherin 23-C Regulates Microtubule Networks by Modifying CAMSAP3's Function.

Cadherin-related 23 (CDH23) is an adhesive protein important for hearing and vision, while CAMSAP3/Marshalin is a microtubule (MT) minus-end binding protein that regulates MT networks. Although both CDH23 and CAMSAP3/Marshalin are expressed in the organ of Corti, and carry several protein-protein interaction domains, no functional connection between these two proteins has been proposed. In this report, we demonstrate that the C isoform of CDH23 (CDH23-C) directly binds to CAMSAP3/Marshalin and modifies its function by inhibiting CAMSAP3/Marshalin-induced bundle formation, a process that requires a tubulin-binding domain called CKK. We further identified a conserved N-terminal region of CDH23-C that binds to the CKK domain. This CKK binding motif (CBM) is adjacent to the domain that interacts with harmonin, a binding partner of CDH23 implicated in deafness. Because the human Usher Syndrome 1D-associated mutation, CDH23 R3175H, maps to the CBM, we created a matched mutation in mouse CDH23-C at R55H. Both in vivo and in vitro assays decreased the ability of CDH23-C to interact with CAMSAP3/Marshalin, indicating that the interaction between CDH23 and CAMSAP3/Marshalin plays a vital role in hearing and vision. Together, our data suggest that CDH23-C is a CAMSAP3/Marshalin-binding protein that can modify MT networks indirectly through its interaction with CAMSAP3/Marshalin.

Indole based Tubulin Polymerization Inhibitors: An Update on Recent Developments

The exploration of cancer microenvironment and its physiology have exposed a number of potential molecular targets for selective therapeutic intervention by anti-cancer agents. Microtubules are basic cell components formed by polymerization of αβ heterodimers which play a pivotal role in cellular functions as well as cell division. Drugs that can control the microtubule assembly either by hindering tubulin polymerization or by obstructing microtubule disassembly are of great importance in anti-cancer therapy. Diverse classes of naturally occurring as well as synthetic and semi-synthetic compounds with an indole nucleus induce microtubule polymerization and depolymerization and thereby change tubulin dynamics. Rapid development of several novel tubulin polymerization inhibitors has been observed over the past few years and some of them have associated vascular disrupting properties too. The present review starts with the structure, function and importance of microtubules in a eukaryotic cell. The well characterized tubulin binding domains and the corresponding inhibitors including their mechanism of action is also a part of this article. The report mainly focuses on the brief synthetic methodology with the relevant SAR studies of different indole derived molecules that have been reported in the past few years as potential inhibitors of tubulin polymerization is discussed. This review will provide the up-to-date evidence-base for synthetic chemists as well as biologists to design and synthesize new active molecules to inhibit tubulin polymerization.

Cytoskeletal Linker Protein Dystonin Is Not Critical to Terminal Oligodendrocyte Differentiation or CNS Myelination.

Oligodendrocyte differentiation and central nervous system myelination require massive reorganization of the oligodendrocyte cytoskeleton. Loss of specific actin- and tubulin-organizing factors can lead to impaired morphological and/or molecular differentiation of oligodendrocytes, resulting in a subsequent loss of myelination. Dystonin is a cytoskeletal linker protein with both actin- and tubulin-binding domains. Loss of function of this protein results in a sensory neuropathy called Hereditary Sensory Autonomic Neuropathy VI in humans and dystonia musculorum in mice. This disease presents with severe ataxia, dystonic muscle and is ultimately fatal early in life. While loss of the neuronal isoforms of dystonin primarily leads to sensory neuron degeneration, it has also been shown that peripheral myelination is compromised due to intrinsic Schwann cell differentiation abnormalities. The role of this cytoskeletal linker in oligodendrocytes, however, remains unclear. We sought to determine the effects of the loss of neuronal dystonin on oligodendrocyte differentiation and central myelination. To address this, primary oligodendrocytes were isolated from a severe model of dystonia musculorum, Dstdt-27J, and assessed for morphological and molecular differentiation capacity. No defects could be discerned in the differentiation of Dstdt-27J oligodendrocytes relative to oligodendrocytes from wild-type littermates. Survival was also compared between Dstdt-27J and wild-type oligodendrocytes, revealing no significant difference. Using a recently developed migration assay, we further analysed the ability of primary oligodendrocyte progenitor cell motility, and found that Dstdt-27J oligodendrocyte progenitor cells were able to migrate normally. Finally, in vivo analysis of oligodendrocyte myelination was done in phenotype-stage optic nerve, cerebral cortex and spinal cord. The density of myelinated axons and g-ratios of Dstdt-27J optic nerves was normal, as was myelin basic protein expression in both cerebral cortex and spinal cord. Together these data suggest that, unlike Schwann cells, oligodendrocytes do not have an intrinsic requirement for neuronal dystonin for differentiation and myelination.

Affinity Purification and Characterization of Functional Tubulin from Cell Suspension Cultures of Arabidopsis and Tobacco.

Microtubules assemble into several distinct arrays that play important roles in cell division and cell morphogenesis. To decipher the mechanisms that regulate the dynamics and organization of this versatile cytoskeletal component, it is essential to establish in vitro assays that use functional tubulin. Although plant tubulin has been purified previously from protoplasts by reversible taxol-induced polymerization, a simple and efficient purification method has yet to be developed. Here, we used a Tumor Overexpressed Gene (TOG) column, in which the tubulin-binding domains of a yeast (Saccharomyces cerevisiae) TOG homolog are immobilized on resin, to isolate functional plant tubulin. We found that several hundred micrograms of pure tubulin can readily be purified from cell suspension cultures of tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). The tubulin purified by the TOG column showed high assembly competence, partly because of low levels of polymerization-inhibitory phosphorylation of α-tubulin. Compared with porcine brain tubulin, Arabidopsis tubulin is highly dynamic in vitro at both the plus and minus ends, exhibiting faster shrinkage rates and more frequent catastrophe events, and exhibits frequent spontaneous nucleation. Furthermore, our study shows that an internal histidine tag in α-tubulin can be used to prepare particular isotypes and specifically engineered versions of α-tubulin. In contrast to previous studies of plant tubulin, our mass spectrometry and immunoblot analyses failed to detect posttranslational modification of the isolated Arabidopsis tubulin or detected only low levels of posttranslational modification. This novel technology can be used to prepare assembly-competent, highly dynamic pure tubulin from plant cell cultures.

Nuclear import mechanism of neurofibromin for localization on the spindle and function in chromosome congression.

Neurofibromatosis type-1 (NF-1) is caused by mutations in the tumor suppressor gene NF1; its protein product neurofibromin is a RasGTPase-activating protein, a property that has yet to explain aneuploidy, most often observed in astrocytes in NF-1. Here, we provide a mechanistic model for the regulated nuclear import of neurofibromin during the cell cycle and for a role in chromosome congression. Specifically, we demonstrate that neurofibromin, phosphorylated on Ser2808, a residue adjacent to a nuclear localization signal in the C-terminal domain (CTD), by Protein Kinase C-epsilon (PKC-ε), accumulates in a Ran-dependent manner and through binding to lamin in the nucleus at G2 in glioblastoma cells. Furthermore, we identify CTD as a tubulin-binding domain and show that a phosphomimetic substitution of its Ser2808 results in a predominantly nuclear localization. Confocal analysis shows that endogenous neurofibromin localizes on the centrosomes at interphase, as well as on the mitotic spindle, through direct associations with tubulins, in glioblastoma cells and primary astrocytes. More importantly, analysis of mitotic phenotypes after siRNA-mediated depletion shows that acute loss of this tumor suppressor protein leads to aberrant chromosome congression at the metaphase plate. Therefore, neurofibromin protein abundance and nuclear import are mechanistically linked to an error-free chromosome congression. Concerned with neurofibromin's, a tumor suppressor, mechanism of action, we demonstrate in astrocytic cells that its synthesis, phosphorylation by Protein Kinase C-ε on Ser2808 (a residue adjacent to a nuclear localization sequence), and nuclear import are cell cycle-dependent, being maximal at G2. During mitosis, neurofibromin is an integral part of the spindle, while its depletion leads to aberrant chromosome congression, possibly explaining the development of chromosomal instability in Neurofibromatosis type-1. Read the Editorial Highlight for this article on page 11. Cover Image for this issue: doi: 10.1111/jnc.13300.

ON THE NATURE OF THE TAU R406W MUTATION: INCREASED CIS PSER404-PRO405 AMIDE BOND IN TAU R406W DUE TO A PHOSPHORYLATION-SENSITIVE CIS-PROLINE-AROMATIC INTERACTION

Tau aggregation as the neurofibrillary tangles observed in Alzheimer's disease is associated with phosphorylation-induced changes in tau structure that promote protein aggregation. The N-terminus and C-terminus of tau interact with the hydrophobic tubulin-binding domains (TBDs), stabilizing their structure. The proline-rich domain is between the N-terminus and TBDs and serves as a linker that allows their dynamic interaction. Phosphorylation and O-GlcNAcylation have opposing functional effects on tau, with phosphorylation promoting tau aggregation and O-GlcNAcylation opposing tau aggregation. At the C-terminus, the R406W mutation accelerates tau aggregation, but the mechanistic basis of this observation is not well understood. Residue 406 of tau follows Ser404-Pro405, with Ser404 phosphorylation observed pathologically and in animal models of Alzheimer's disease. The prolyl isomerase Pin1, which binds and isomerizes pSer-Pro sequences, implicates prolyl isomerization in misfolding of hyperphosphorylated tau. The structural effects of phosphorylation versus O-GlcNAcylation were examined in the tau proline-rich domain and in the C-terminal domain by circular dichroism and heteronuclear NMR spectroscopy via examination of non-phosphorylated, phosphorylated, and O-GlcNAcylated peptides comprising tau174-251 and tau395-411. Peptides were also examined with the R406W mutation in both non-phosphorylated and phosphorylated forms. Phosphorylation was found to induce a strong disorder-to-order transition in the proline-rich domain, with special ordering observed on the residues pT175, pT181, pT212, pT217, and pT231. Phosphothreonine adopts a stable, ordered structure with a strong hydrogen bond to its own amide. O-GlcNAcylation opposed this structural change. We hypothesized that mutation of R406 to tryptophan (R406W) would lead to increased cis amide bond. R406W tau peptides exhibited increased population of cis amide bond, due to a favorable H a -cis-Pro-aromatic C-H/p interaction. Phosphorylation of Ser404 further increased the population of cis-proline at pSer404-Pro405, with greatest effect in R406W. These data suggest a disorder-to-order transition upon phosphorylation in the proline-rich domain as a basis for changes in the dynamic structure of tau. R406W mutation results in higher populations of cis amide bond. Phosphorylation of Ser404 leads to further increases in population of cis amide bond, particularly in R406W. Cis amide bond prevents phosphatase activity, and therefore tau-R406W induces both greater structural changes and longer phosphorylation residence times.

A novel neurofibromin (NF1) interaction with the leucine‐rich pentatricopeptide repeat motif‐containing protein links neurofibromatosis type 1 and the french canadian variant of leigh's syndrome in a common molecular complex

Loss-of-function mutations and deletions in the neurofibromin tumor suppressor gene (NF1) cause neurofibromatosis type 1 (NF-1), the most common inherited syndrome of the nervous system in humans, with a birth incidence of 1:3,000. The most visible features of NF-1 are the neoplastic manifestations caused by the loss of Ras-GTPase-activating protein (Ras-GAP) activity mediated through the GAP-related domain (GRD) of neurofibromin (NF1), the protein encoded by NF1. However, the syndrome is also characterized by cognitive dysfunction and a number of developmental abnormalities. The molecular etiology of many of these nonneoplastic phenotypes remains unknown. Here we show that the tubulin-binding domain (TBD) of NF1 is a binding partner of the leucine-rich pentatricopeptide repeat motif-containing (LRPPRC) protein. These two proteins complex with Kinesin 5B, hnRNP A2, Staufen1, and Myelin Basic Protein (MBP) mRNA, likely in RNA granules. This interaction is of interest in that it links NF-1 with Leigh's syndrome, French Canadian variant (LSFC), an autosomal recessive neurodegenerative disorder that arises from mutations in the LRPPRC gene. Our findings provide clues to how loss or mutation of NF1 and LRPPRC may contribute to the manifestations of NF-1 and LSFC.

Disease Associated Mutations in Tau Alter its Interactions with Tubulin

The intrinsically disordered protein tau forms filamentous aggregates in association with a variety of neurodegenerative disorders, including Alzheimer's disease. Functionally, tau binds to both soluble tubulin and microtubules, and enhances microtubule assembly. In order to probe the relationship between functional and disease-associated interactions of tau, we made two point mutations to tau in its microtubule binding region, which also forms the core aggregated domain in disease: P301L, which is associated with frontotemporal dementia, and P332L, a designed mutant. A combination of fluorescence correlation spectroscopy (FCS) and single molecule Forster resonant energy transfer (FRET) was used to characterize the interaction of wild-type and mutant tau with tubulin. We find that both proline mutants have enhanced affinity for tubulin, which we believe may be due to an increased amount of α-helical structure in the tubulin-binding domain of tau. Interestingly, we observe that the increased affinity for tubulin is inversely correlated with the ability to enhance microtubule assembly. Our results suggest that some disease-associated mutations to tau may alter the balance of tubulin binding and microtubule assembly that is necessary for maintaining a dynamic microtubule cytoskeleton.